The present invention relates to polypeptides, polynucleotides and uses thereof, in particular to members of the stress-activated protein kinase (SAPK) family.
Four mitogen-activated protein (MAP) kinase family members are activated by cellular stresses (chemical, heat and osmotic shock, ultraviolet radiation, inhibitors of protein synthesis), bacterial lipopolysaccharide (LPS), and the cytokines interleukin-1 (IL1) and tumour necrosis factor (TNF), and have therefore been termed stress-activated protein kinases or SAPKs (reviewed in Cohen, 1997). Isoforms of SAPK1 [also called c-Jun N-terminal kinases (JNKs)] phosphorylate Ser-63 and Ser-73 in the activation domain of c-Jun (Pulverer et al, 1991), thereby increasing its transcriptional activity. The transcription factors Elk1 (Cavigelli et al, 1995 and ATF2 are phosphorylated in vitro (the latter at Thr-69, Thr-71, and Ser-90 (Gupta et al, 1995; Livingstone et al, 1995), increasing the transcriptional activity of these proteins. The same sites in the transcription factor c-Jun also become phosphorylated when cells are exposed to the stresses and cytokines that activate SAPK1, or after cotransfection with protein kinases known to activate SAPK1 (Pulverer et al, 1991; Hibi et al, 1993; Dxc3xa9rijard et al, 1994; Kyriakis et al, 1994; Cavigelli et al, 1995; Gupta et al, 1995; Whitmarsh et al, 1995; Zinck et al, 1995), suggesting that c-Jun is a physiological substrate for SAPK1.
SAPK2a [also termed p38 (Han et al, 1994), p40 (Freshney et al, 1994), RK (Rouse et al, 1994), CSBP (Lee et al, 1994) and Mxi2 (Zervos et al, 1995)] is inhibited very specifically by the pyridinyl imidazoles SB 203580 and SB 202190 (Lee et al, 1994; Cuenda et al., 1995; reviewed in Cohen, 1997) which have been exploited to identify several physiological substrates. These include four protein kinases, namely MAP kinase-activated protein kinase-2 (MAPKAP-K2, Rouse et al, 1994) and the closely related MAPKAP-K3, which shares 75% amino acid sequence identity and has similar substrate specificity in vitro (McLaughlin et al, 1996; Ludwig et al, 1996; Clifton et al, 1996), as well as MAP kinase interacting protein kinases-1 and -2 (Mnk1 and Mnk2) (Waskiewicz et al, 1997; Fukunaga and Hunter, 1997).
Physiological substrates of MAPKAP-K2/K3 include heat shock protein (HSP) 27 (Stokoe et al, 1992a; Cuenda et al, 1995; Huot et al, 1995) and the transcription factor CREB (Tan et al, 1996), whereas transcription factor eIEF4E is a physiological substrate of Mnk1/2 (Waskiewicz et al, 1997). The phosphorylation of HSP27 appears to enhance the polymerisation of actin and is thought to help repair the actin microfilament network which becomes disrupted during cellular stress, thereby aiding cell survival (Lavoie et al, 1995). The phosphorylation of CREB (at Ser-133) is essential to allow this protein to stimulate the ascription of genes that contain cyclic AMP response elements (CREs). SAPK2a also mediates the stress-induced phosphorylation (at Ser-78 and Ser-81) and activation of the CEBPxcex2-related transcription factor CHOP (Wang and Ron, 1996) and the ternary complex factor Elk-1 (Price et al, 1996).
Based on the effects of SB 203580, the activation of SAPK2a is rate-limiting in the LPS-induced production of IL1 and TNF in monocytes (Lee et al, 1994), in the TNF-stimulated transcription of IL6 and GM-CSF in fibroblasts (Beyaert et al, 1996), in the IL1-induced stimulation of glucose uptake in epithelial cells (Gould et al, 1995), in collagen-induced platelet aggregation (Saklatvala et al, 1996), in the stress-induced transcription of c-Jun and c-Fos in fibroblasts (Hazzalin et al, 1996; Price et al, 1996) and in the LPS-induced synthesis of cyclo-oxygenase-2 (COX-2, the rate limiting enzyme in prostaglandin synthesis) in RAW264.7 macrophages (Paul et al 1996). Since the c-fos and COX2 promoters contain CREs, the MAPKAP-K2/K3 mediated phosphorylation of CREB may contribute to the stress and cytokine-induced transcription of these two genes The importance of the CRE in the induction of c-fos mRNA is well documented (Ginty et al 1994). The SAPK2a catalysed phosphorylation of Elk-1 (Price et al, 1996) and the MAPKAP-K2 catalysed phosphorylation of CREB (Tan et al, 1996) are both likely to contribute to the stress-induced transcription of c-fos (Ginty et al, 1994). The transcription factors ATF2 and Elk-1 are also phosphorylated by SAPK2 in vitro and after transfection of mammalian cells with the upstream activators of SAPK2 that do not activate SAPK1 (Raingeaud et al, 1996).
Recently, two additional SAP kinases were identified, called SAPK2b [or p38xcex2 (Jiang et al, 1996)] and SAPK3 (Mertens et al, 1996) [also called ERK6 (Lechner et al, 1996) and p38xcex3 (Li et al, 1996)]. The amino acid sequence of SAPK2b is 73% identical to SAPK2a and it is inhibited by SB 202190 at similar concentrations to SAPK2a. In contrast, the amino acid sequence of SAPK3 is only 60% identical to SAPK2a and SAPK2b and 47% identical to SAPK1. Like SAPK2, SAPK3 contains a TGY motif in the activation domain (which is TPY in SAPK1 and TEY in p42 and p44 MAP kinases) and subdomain VII is separated by six amino acids from the activation loop in subdomain VIII (as compared to eight residues in SAPK1 and  greater than 12 residues in any other MAP kinase family member). SAPK2b has been introduced into mammalian cells by transient transfection and shown to be activated in response to pro-inflammatory cytokines and stressful stimuli in a manner similar to SAPK1 and SAPK2a. The physiological roles of SAPK2b and SAPK3 are unknown. The mRNAs encoding these enzymes are present in all mammalian tissues examined (Jiang et al, 1996; Mertens et al, 1996; Goedert et al., 1997), with the mRNA encoding SAPK3 being highest in skeletal muscle. Expression of wild-type SAPK3 and an inactive mutant in the muscle cell line C2C12 enhanced and inhibited differentiation into myotubes, respectively (Lechner et al, 1996). In vitro, SAPK2b and SAPK3 phosphorylated several proteins that are also substrates for SAPK2a. SAPK2b was reported to phosphorylate the transcription factor ATF2 more efficiently than SAPK2a (Jiang et al., 1996) but, since the stress- and cytokine-induced phosphorylation of ATF2 in fibroblasts is unaffected by SB 203580 (Hazzalin et al., 1996; Beyaert et al., 1996), neither SAPK2a nor SAPK2b appears to be rate-limiting for ATF2 phosphorylation in vivo, in contrast to earlier studies using transfection-based approaches (Gupta et al, 1995). However, whether SAPK1 and/or SAPK3 are rate-limiting for ATF2 phosphorylation in vivo is unknown.
Five chromatographically distinct SAP kinase kinases (SKKs or SAPKKs) have been identified in mammalian cells (Meier et al, 1996; Cuenda et al, 1996). In vitro, SKK1 [also termed MKK4 (Dxc3xa9rijard et al, 1995), SEK1 (Sanchez et al, 1994) and XMEK2 (Yashar et al, 1993)] activates all four SAPKs 1, 2a and 2b (Sanchez et al, 1994; Dxc3xa9rijard et al, 1995; Doza et al, 1995; Jiang et al, 1996), although SAPK2b is phosphorylated less efficiently. SKK2 [also termed MKK3 (Dxc3xa9rijard et al, 1995)] and SKK3 (Cuenda et al, 1996) [also called MKK6 (Han et al, 1996; Moriguchi et al, 1996; Raingeaud et al, 1996) and MEK6 (Stein et al, 1996)] activate SAPK2a but not SAPK1. SKK3 was also the most efficient activator of SAPK2b in co-transfection experiments (Jiang et al, 1996). SKK4 and SKK5 activate SAPK1 but not SAPK2a (Meier et al, 1996). SKK4/SKK5 have not been purified or cloned and their amino acid sequences are thus still unknown. SKK1 and SKK2 are the only activators of SAPK2 generated when rat pheochromocytoma (PC12) cells are exposed to chemical stress, osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin (Meier et al, 1996). However, SKK3 is the dominant activator of SAPK2 and SKK4/SKK5 is/are the dominant activator(s) of SAPK1 when human epithelial (KB) cells are exposed to the same stresses as PC12 cells or stimulated with IL-1, or when human (THP1) monocytes are stimulated with LPS (Meier et al, 1996; Cuenda et al, 1996). The identity of the upstream activators of SAPK1 and SAPK2 may therefore vary from cell to cell.
At least five enzymes capable of activating SKK1, SKK2 and SKK3 in vitro and/or in cotransfection experiments have been identified, namely MEK kinase (MEKK) (Yan et al, 1994; Lin et al, 1995; Matsuda et al, 1995; Blank et al, 1996), MAP kinase upstream kinase (MUK) (Hirai et al, 1996), mixed lineage kinase-3 (MLK3) (Rana et al, 1996), TGFb-activated protein kinase-1 (TAK-1) (Moriguchi et al, 1996) and the protooncogene Tp12 (Salmeron et al, 1996) MEKK activates cotransfected SAPK1 more effectively than cotransfected SAPK2 or MAP kinase kinase-1 (MKK1, a physiological activator of p42 and of 44 MAP kinases) (Xu et al,1996). However, SKK1 can also be activated by MUK, MLK3 and Tp12, SKK2 by TAK1, while Tp12 can also activate MAPKK1. Further work is needed to understand which (if any) of these kinases activate each SAPKK in vivo under different conditions.
We here report the cloning of a novel MAP kinase family member that we call SAPK4, and characterisation of SAPK3 and SAPK4. SAPK4, which also contains a TGY sequence in the activation domain, shows about 60% identity to SAPK2a, SAPK2b and SAPK3 and its mRNA is widely expressed in human tissues. We show that SAPK4 is activated by the same stimuli that activate other SAP kinases and that SKK3 is likely to be the major upstream activator of SAPK3 and SAPK4 in vivo. The substrate specificity of SAPK4 in vitro is similar to that of SAPK3 and neither SAPK3 nor SAPK4 are inhibited by SB 203580 or SB 202190.
A first aspect of the invention provides a substantially pure stress-activated protein kinase comprising the amino acid sequence identified as SEQ. ID. NO: 1.
or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative.
The polypeptide with the amino acid sequence as shown above is herein referred to as SAPK4 (stress-activated protein kinase 4).
By xe2x80x9csubstantially purexe2x80x9d we mean that the said protein kinase is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said protein kinase, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said protein kinase.
Thus, the invention also includes compositions comprising the said protein kinase and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.
The invention also includes the substantially pure said protein kinase when combined with other components ex vivo, said other components not being all of the components found in the cell in which said protein kinase is found.
By xe2x80x9cvariantsxe2x80x9d of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said protein kinase.
By xe2x80x9cconservative substitutionsxe2x80x9d is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
It is particularly preferred if the stress-activated protein kinase variant has an amino acid sequence which has at least 65% identity with the amino acid sequence given above, more preferably at least 70%, still more preferably at least 75%, yet still more preferably at least 80%, in further preference at least 85%, in still further preference at least 90% and most preferably at least 95% or 97% identity with the amino acid sequence given above.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
A particular embodiment of the invention provides a substantially pure human SAPK4 polypeptide which consists of the amino acid sequence identified as SEQ. ID. NO: 1.
or naturally occurring allelic variants thereof.
It is particularly preferred, although not essential, that the variant or fragment or derivative or fusion of the said protein kinase, or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of SAPK4 with respect to the phosphorylation of myelin basic protein. It is more preferred if the variant or fragment or derivative or fusion of the said protein kinase, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the enzyme activity of SAPK4 with respect to the phosphorylation of myelin basic protein. However, it will be appreciated that variants or fusions or derivatives or fragments which are devoid of enzymatic activity may nevertheless be useful, for example by interacting with another polypeptide.
A second aspect of the invention provides a recombinant polynucleotide encoding a protein kinase as defined in the first aspect of the invention or encoding a variant or fragment or derivative of fusion of said kinase or a fusion of a said variant or fragment or derivative. Preferences for the said protein kinase variant are the same as in the first aspect of the invention.
In one preferred embodiment the polynucleotide comprises the nucleotide sequence identified as SEQ. ID. NO: 2 as follows.
or a variant, fragment, fusion or derivative thereof.
The invention also includes a polynucleotide comprising a fragment of the recombinant polynucleotide of the second aspect of the invention. Preferably, the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length. Such polynucleotides are useful as PCR primers.
The polynucleotide or recombinant polynucleotide may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.
A xe2x80x9cvariationxe2x80x9d of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle; xe2x80x9cStrategies and Applications of In Vitro Mutagenesis,xe2x80x9d Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, the polynucleotide of the invention includes polynucleotide that shows at least 55 per cent, preferably 60 per cent, and more preferably at least 70 per cent and most preferably at least 90 per cent homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.
DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1xc3x97SSC and 6xc3x97SSC and at temperatures of between 55xc2x0 C. and 70xc2x0 C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By xe2x80x9chigh stringencyxe2x80x9d we mean 2xc3x97SSC and 65xc2x0 C. 1xc3x97SSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.
xe2x80x9cVariationsxe2x80x9d of the polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 80% and preferably at least 90 or 95%) with equivalent stretches of the polynucleotide of the invention even though the overall homology between the two polynucleotides may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.
A further aspect of the invention provides a replicable vector comprising a recombinant polynucleotide encoding a said protein kinase or a variant, fragment, derivative or fusion of said protein kinase or a fusion of said variant, fragment or derivative.
A variety of methods have been developed to operably link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3xe2x80x2-single-stranded termini with their 3xe2x80x2-5xe2x80x2-exonucleolytic activities, and fill in recessed 3xe2x80x2-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487491. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
In this method the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention. Thus, the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in U.S. Pat. No. 4,440,859 issued Apr. 3, 1984 to Rutter et al, U.S. Pat. No. 4,530,901 issued Jul. 23, 1985 to Weissman, U.S. Pat. No. 4,582,800 issued Apr. 15, 1986 to Crowl, U.S. Pat. No.4,677,063 issued Jun. 30, 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued Jul. 7, 1987 to Goeddel, U.S. Pat. No. 4,704,362 issued Nov. 3,1987 to Itakura et al, U.S. Pat. No. 4,710,463 issued Dec. 1, 1987 to Murray, U.S. Pat. No. 4,757,006 issued Jul. 12, 1988 to Toole, Jr. et al, U.S. Pat. No. 4,766,075 issued Aug. 23, 1988 to Goeddel et al and U.S. Pat. No. 4,810,648 issued Mar. 7, 1989 to Stalker, all of which are incorporated herein by reference.
The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, anital cells and insect cells.
The vectors include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells; such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA.
Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.
For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5xc3x97PEB using 6250V per cm at 25 xcexcFD.
Methods for transformation of yeast by electroporation are disclosed in Becker and Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.
In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
A further aspect of the invention provides a method of making a stress-activated protein kinase or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said stress-activated protein kinase, and isolating said protein kinase or a variant, derivative, fragment or fusion thereof of a fusion of a variant, fragment or derivative from said host cell. Methods of cultivating host cells and isolating recombinant proteins are well known in the art.
The invention also includes a stress-activated protein kinase, or a variant, fragment, derivative or fusion thereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention.
A still further aspect of the invention provides an antibody reactive towards a stress-activated protein kinase of the invention.
Antibodies reactive towards the said stress-activated protein kinase of the invention may be made by methods well known in the art. In particular, the antibodies may be polyclonal or monoclonal.
Suitable monoclonal antibodies which are reactive towards the said protein kinase may be prepared by known techniques, for example those disclosed in xe2x80x9cMonoclonal Antibodies: A mammal of techniquesxe2x80x9d, H Zola (CRC Press, 1988) and in xe2x80x9cMonoclonal Hybridoma Antibodies: Techniques and Applicationsxe2x80x9d, SGR Hurrell (CRC Press, 1982).
In a preferred embodiment the antibody is raised using any suitable peptide sequence obtainable from the given amino acid sequence of SAPK4. It is preferred if polyclonal antipeptide antibodies are made.
It is particularly preferred if the antibody does not react substantially with another stress-activated protein kinase such as SAPK2a, SAPK2b or SAPK3. Accordingly, it may be preferred if peptides based on the SAPK4 sequence are used which vary significantly from any peptides found in any other stress-activated protein kinases such as SAPK2a, SAPK2b or SAPK3.
Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised, may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged. Such modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism. The peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the peptide to be formed as a loop, with the N-terminal and C-terminal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the peptide is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the peptide of the invention forms a loop.
According to current immunological theories, a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope. The peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin. More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1. The latter compound may variously be regarded as a carrier or as an adjuvant or as both. Alternatively, several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking. Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, the latter agent exploiting the xe2x80x94SH group on the C-terminal cysteine residue (if present).
If the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen""s xe2x80x9cEcosecxe2x80x9d system is an example of such an arrangement. The peptide of the invention may be linked to other antigens to provide a dual effect.
Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4xe2x80x2-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG72QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
A further aspect of the invention provides a method of identifying a compound that inhibits the activity of a stress-activated protein kinase as defined in the first aspect of the invention or of the stress-activated protein kinase SAPK3, the method comprising contacting a compound with the stress-activated protein kinase or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the activity of the said is protein kinase is reduced compared to the activity of the said protein kinase or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.
It will be appreciated that SAPK3 is a stress-activated protein kinase with a similar substrate specificity to SAPK4 (Cuenda et al (1997); Example 1 and Example 4). Conveniently, the method makes use of the fact that SAPK3 or SAPK4 phosphorylates myelin basic protein as described in Example 1 and Example 4, but any suitable substrate may be used. For example, Elk-1, ATF2, SAP-1, MAPKAP-K2, MAPKAP-K3, p53, SAP-2 or c-jun may be used as a substrate; Elk-1, ATF2 or SAP-1 are particularly preferred.
Conveniently, the method makes use of an assay which may be substantially the same as that disclosed in Example 4 and Cuenda et al (1997) EMBO J. 16, 295-305, incorporated herein by reference, except that SAPK3 may be replaced by SAPK4.
A still further aspect of the invention provides a method of identifying a compound which blocks the activation of the said stress-activated protein kinase as defined in the first aspect of the invention or the stress-activated protein kinase SAPK3 by SKK3 the method comprising determining whether a compound enhances or disrupts the interaction between (a) a stress-activated protein kinase as defined in the first aspect of the invention or SAPK3 or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) SKK3 or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said stress-activated protein kinase or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by SKK3 or a suitable variant, derivative, fragment or fusion thereof.
Activation of SAPK3 or SAPK4 by SKK3 or SKK1 may be assayed substantially as described in Example 4 or Cuenda et al (1997) EMBO J. 16, 295-305, incorporated herein by reference, except that SAPK3 may be replaced by SAPK4.
By xe2x80x9cSKK3xe2x80x9d we mean stress-activated protein kinase kinase 3 (SAP kinase kinase 3; SAPKK3) as described in Example 1. SKK1 may also be used and so the invention also includes a method of identifying a compound which blocks the activation of the said stress-activated protein as defined in the first aspect of the invention by SKK1.
Conveniently, the said stress-activated protein kinase or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology. Similarly, it is preferred if the SKK3 or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology.
It is also preferred if SKK3 is SKK3 purified from an appropriate natural source; for example, SKK3 may be purified using the method described in Cuenda et al (1996) EMBO J. 15, 4156-4164.
It will be appreciated that by xe2x80x9csuitablexe2x80x9d we mean that the said components in the method are those that have interactions or activities which are substantially the same as those of SAPK3 or SAPK4 or SKK3 as the case may be but which may be more convenient to use in an assay. For example, fusions of SAPK3 or SAPK4 or SKK3 are particularly useful since fusion may contain a moiety which may allow the fusion to be purified readily.
The enhancement or disruption of the interaction between the said stress-activated protein kinase and SKK3, or suitable derivatives, fragments, fusions or variants can be measured in vitro using methods well known in the art of biochemistry and include any methods which can be used to assess protein-protein interactions.
The said interaction can also be measured within a cell, for example using the yeast two hybrid system as is well known in the art.
It will be appreciated that the invention provides screening assays for drugs which may be useful in modulating the activity of SAPK3 or SAPK4 or their interactions with SKK3. The compounds identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds.
A further aspect of the invention provides a compound identifiable by the screening methods of the invention. A still further aspect provides such a compound for use in medicine.
It is believed that such compounds are useful in treating inflammatory disease. Inflammatory diseases include rheumatoid arthritis, psoriasis, septic shock, asthma and inflammatory bowel disease.
Thus, a further aspect of the invention provides a method of treating a patient with an inflammatory disease the method comprising administering to the patient an effective amount of a compound identifiable by the screening methods of the invention.
A still further invention provides a use of a compound identifiable by the screening methods of the invention in the manufacture of a medicament for treating an inflammatory disease in a patient.
The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (e.g. subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.
Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. Thus, the invention also provides pharmaceutical compositions comprising the compound identifiable by the screening methods of the invention and a pharmaceutically acceptable carrier.
Further aspects of the invention provide a use of a stress-activated protein kinase or a variant, fragment, derivative or fusion thereof or a fusion of a said variant, derivative or fragment as defined in the first aspect of the invention in a screening assay for compounds which inhibit the activity of the said protein kinase or which block the activation of said protein kinase by SKK3.
A further aspect of the invention provides a kit of parts that are useful in carrying out the screening methods.
A still further aspect of the invention provides the use of SAPK3 (in vitro or in vivo), its analogues, isoforms, inhibitors, activators and the functional equivalents thereof in the regulation of protein phosphorylation, gene expression and/or protein synthesis. A composition comprising such agents is also covered by the present invention, and the use of such a composition for treatment of disease states where protein phosphorylation, gene expression and/or protein synthesis exhibit abnormality is also provided.
The invention also provides the use of SAPK3 in the manufacture of a medicament for the treatment of disease states where protein phosphorylation, gene expression and/or protein synthesis exhibit abnormality.
Also provided is a method (in vitro or in vivo) of identifying agents able to influence the activity of SAPK3, said method comprising:
a. exposing a test substance to SAPK3 or a functional equivalent thereof in the presence of a substrate for SAPK3;
b. detecting whether (and, optionally, to what extent) said substrate has been phosphorylated.
Detection of the presence and/or amount of phosphate associated with or dissociated from the substrate after the assay may be conducted by any convenient means. Generally detection may be accomplished by using labelled (e.g. radiolabelled) phosphate in free solution or attached to the substrate, and comparing the amount associated with (or dissociated from) the substrate before and after the assay.
The test substance may be an analogue, isoform, inhibitor, or activator of SAPK3.
The substrate can comprise myelin basic protein (MBP), or a transcription factor such as ATF-2. In certain embodiments the test substance can itself act as a substrate, and can be capable of being phosphorylated. Thus, the substrate can be a phosphatase which is itself capable of affecting protein phosphorylation, gene expression and/or protein synthesis.
Some of the assay components may be localised on a surface, such as a blotting membrane, or an assay plate for ELISA etc, although the assay can be carried out in solution.
A further aspect of the invention is the use of SKK3 in a method of identifying agents able to influence the activity of a stress-activated protein kinase or a variant, fragment, derivative or fusion thereof or a fusion of a said variant, derivative or fragment as defined in the first aspect of the invention (SAPK4) or SAPK3 or a variant, fragment, derivative or fusion thereof or a fusion of a said variant, derivative or fragment thereof (SAPK3), said method comprising:
a) exposing SAPK4 or SAPK3 to SKK3;
b) exposing a test substance to SAPK4 or SAPK3 in the presence of a substrate for SAPK4 or SAPK3 respectively;
c) detecting whether (and, optionally, to what extent) said substrate has been phosphorylated.
A still further aspect is a method of activating a stress-activated protein kinase or a variant, fragment, derivative or fusion thereof or a fusion of a said variant, derivative or fragment as defined in Claim 1 (SAPK4) or SAPK3 or a variant, fragment, derivative or fusion thereof or a fusion of s a said variant, derivative or fragment thereof (SAPK3), said method comprising incububating SAPK4 or SAPK3 with SKK3.
It will be appreciated that SKK1 and SKK3 may be functional equivalents with regard to activating SAPK3 and SAPK4, and that SKK1 may be used instead of SKK3. It is preferred that SKK3 is used.
It will be appreciated that the SKK3 or SKK1 may be purified; preferably the SKK3 or SKK1 is purified. It will further be appreciated that the SKK3 or SKK1 may be recombinant.
In a further aspect the present invention provides a method of treatment of the human or non-human (preferably mammalian) animal body, said method comprising administering SAPK3, its analogues, inhibitors, stimulators or functional equivalents thereof to said body. Said method affects the regulation of gene expression in the treated body, preferably by affecting the activity of gene transcription factors.
The present invention also provides a therapeutic agent able to affect gene expression and cell regulation by interaction with SAPK3, its analogues, inhibitors, stimulators or functional equivalents. Such therapeutic agents are likely to be efficacious as anti-inflammatory agents, and/or as immunosuppressants, anti-apoptosis and anti-cancer drugs.
Variants (whether naturally-occurring or otherwise) may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below. By xe2x80x9cfragment of said protein kinasexe2x80x9d we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding assay.
By xe2x80x9cfusion of said protein kinasexe2x80x9d we include said protein kinase fused to any other polypeptide. For example, the said protein kinase may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said protein kinase. Similarly, the said protein kinase may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any variant, fragment or derivative of said protein kinase are also included in the scope of the invention.